The field of the invention is cell culture and medical biotechnology, particularly hepatocyte cell cultures used in liver assist devices for treating a patient with liver disease. Hepatocyte cells are induced in vivo, procured from the liver organ, cultured and incorporated in a device to treat a patient via the bloodstream to provide hepatic function. The hepatocyte isolation methods of the invention provide enhanced cell function that extends the functionality of the cells in the course of treating the patient.
Extracorporeal liver assist devices (LAD) have been proposed as a treatment for patients in acute or fulminant liver failure. The LAD would function as a temporary support designed to provide hepatic function until liver transplantation or the regeneration of the patient""s own liver. The LAD incorporates a bioreactor containing isolated porcine hepatocytes that are expected to detoxify substances in the circulating plasma of patients in liver failure. However, one of the challenges in using isolated hepatocytes is that many of these differentiated activities are transient, lasting only hours to a few days in culture (Nishibe, Y, and Hirata, M. Induction of cytochrome P-450 isozymes in cultured monkey hepatocytes. Int J Biochem Cell Bio. 27:3:279-285. 1995. Jauregui, H O, Ng, S F, Gann, K L and Waxman, D J. Xenobiotic induction of P-450 PB-4 (IIB1) and P-450c (IA1) and associated monooxygenase activities in primary cultures of adult rat hepatocytes. Xeno, 21(9):1091-106. 1991. Niak, S, Trenkler, D, Santangini, H, Pan, J and Jauregui, H O. Isolation and culture of porcine hepatocyte for artificial liver support. Cell Trans 5:107-115, 1996.) These functional detoxification activities exist as a family of enzymes, including cytochrome P450 isoenzymes, with each enzyme responsible for metabolism of specific substrates.
While the roles of hepatocytes in a LAD are multifold, one of their most critical functions is detoxification mediated by detoxification enzymes. Therefore, the maintenance of P450 cytochrome and other detoxification activity of hepatocyte cultures is of interest in the successful treatment of fulminant hepatic failure with a liver assist device.
The method of the invention increases enzyme activity in normal hepatocytes as much as 100-fold or more and that enhanced activity is maintained for at least one week in culture. This sustained level of detoxification activity from in vivo induction methods is significantly higher than levels found in non-induced hepatocytes or those obtained using in vitro induction methods. The cells maintain this level of function when incorporated in bioreactor culture to produce cell products and metabolize toxic substances. The invention described here would serve the medical community by increasing the detoxification capabilities of hepatocytes to be used therapeutically when the bioreactor is used as, or incorporated into, a liver assist device.
The invention features a liver cell culture comprising hepatocytes that have increased functional enzyme activity when isolated from a liver of a donor that had been administered at least one induction agent in vivo prior to isolation of hepatocyte cells from the liver. The induced hepatocytes are used in a bioreactor and cultured to produce hepatocyte cell products or metabolize toxins added to the culture, or both. In the preferred embodiment, the bioreactor is, or is an integral part of, a liver assist device used to treat a patient in need of liver assist. In another preferred embodiment at least two cultures of hepatocytes from different isolations induced by different induction agents may be mixed or used together in a bioreactor to have a bioreactor that exhibits a wider range of increased functional enzyme activity.